Carbon and Nitrogen Acquisition in Shallow and Deep Holobionts of the Scleractinian S. pistillata Leïla Ezzat, Maoz Fine, Jean-François Maguer, Renaud Grover, Christine Ferrier-Pagès

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Leïla Ezzat, Maoz Fine, Jean-François Maguer, Renaud Grover, Christine Ferrier-Pagès. Carbon and Nitrogen Acquisition in Shallow and Deep Holobionts of the Scleractinian Coral S. pistillata. Frontiers in Marine Science, Frontiers Media, 2017, 4, pp.UNSP 102. ￿10.3389/fmars.2017.00102￿. ￿hal-02572876￿

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ORIGINAL RESEARCH published: 13 April 2017 doi: 10.3389/fmars.2017.00102

Carbon and Nitrogen Acquisition in Shallow and Deep Holobionts of the Scleractinian Coral S. pistillata

Leïla Ezzat 1*, Maoz Fine 2, 3, Jean-François Maguer 4, Renaud Grover 1 and Christine Ferrier-Pagès 1

1 Marine Department, Scientific Centre of Monaco, Monaco, Monaco, 2 The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel, 3 The Interuniversity Institute for Marine Sciences, Eilat, Israel, 4 Laboratoire de l’Environnement Marin, UMR 6539, UBO/Centre Nationnal de la Recherche Scientifique/IRD/IFREMER, Institut Universitaire Européen de la Mer, Plouzané, France

Reef building can host different symbiont genotypes (clades), and form distinct holobionts in response to environmental changes. Studies on the functional significance of genetically different symbionts have focused on the thermal tolerance rather than on the nutritional significance. Here, we characterized the nitrogen and carbon assimilation rates, the allocation patterns of these nutrients within the symbiosis, and the trophic condition of two distinct holobionts of pistillata: one associated with

Edited by: Symbiodinium clade A in shallow reefs and the other one associated with clade C in Noga Stambler, mesophotic reefs. The main findings are that: (1) clade C-symbionts have a competitive Bar-Ilan University, Israel advantage for the acquisition of carbon at low irradiance compared to clade A-symbionts; Reviewed by: Susana Enríquez, (2) light is however the primary factor that determines the positive relationship between National Autonomous University of the amount of carbon fixed in photosynthesis by the symbionts and the amount of Mexico, Mexico carbon translocated to the host; and (3) by contrast, the dominant Symbiodinium type Michael P. Lesser, University of New Hampshire, USA preferentially determines a negative relationship between rates of coral feeding and David Michael Baker, nitrogen assimilation, although light still plays a relevant role in this relationship. Clade University of Hong Kong, Hong Kong C-holobionts had indeed higher heterotrophic capacities, but lower inorganic nitrogen *Correspondence: Leïla Ezzat assimilation rates than clade A-holobionts, at all light levels. Broadly, our results show leila@centrescientifique.mc that the assimilation and translocation rates of inorganic carbon and nitrogen are clade and light-dependent. In addition, the capacity of S. pistillata to form mesophotic reefs in Specialty section: the Red Sea relies on its ability to select Symbiodinium clade C, as this symbiont type is This article was submitted to Coral Reef Research, more efficient to fix carbon at low light. a section of the journal Frontiers in Marine Science Keywords: light acclimation, carbon isotopes, nitrogen isotopes, clades, nitrate, ammonium, scleractinian coral, mesophotic coral Received: 14 December 2016 Accepted: 27 March 2017 Published: 13 April 2017 INTRODUCTION Citation: Ezzat L, Fine M, Maguer J-F, Grover R Coral reefs support one of the most diverse and productive biological communities on Earth. Their and Ferrier-Pagès C (2017) Carbon success owe from the symbiotic relationship that corals build with photosynthetic dinoflagellate and Nitrogen Acquisition in Shallow and Deep Holobionts of the algae, commonly named zooxanthellae, that can sustain up to 95% of the daily energetic demand of Scleractinian Coral S. pistillata. the symbiotic association (Muscatine et al., 1984; Muscatine, 1990), also called holobiont (Rohwer Front. Mar. Sci. 4:102. et al., 2002). While symbionts need light for photosynthesis and constrain their host to mainly doi: 10.3389/fmars.2017.00102 develop in the first 30m depth, corals can however form large structures called mesophotic reefs

Frontiers in Marine Science | www.frontiersin.org 1 April 2017 | Volume 4 | Article 102 Ezzat et al. Nutrient Acquisition in Mesophotic Corals in much deeper waters. Mesophotic reefs therefore refer to deep heterotrophic capacities of the host (Leal et al., 2015). Overall, but light-limited coral ecosystems (Hinderstein et al., 2010), these studies suggest that symbiont identity directly relates to ranging between 30 and 80m in the Gulf of Mexico (Kahng, the trophic plasticity of the holobiont. Because acquisition of 2010), to more than 100 m in some locations with exceptionally both auto-and heterotrophic nutrients is critical for coral growth clear waters, such as the Red Sea (Lesser et al., 2009; Winters and reproduction (Muscatine and Porter, 1977; Muscatine et al., et al., 2009). Although, photosynthesis is limited by the low light 1989; Ferrier-Pages et al., 2000; Houlbreque and Ferrier-Pagès, levels, these reefs host a wide diversity of taxa due to numerous 2009), a better understanding of the nutritional capacities of upwelling events and internal waves, that deliver significant different holobionts will help predict their resilience to stress. amounts of inorganic nutrients and particulate organic matter Until now, no direct simultaneous comparison exists between (Leichter and Genovese, 2006; Lesser et al., 2009). auto-and heterotrophic performance of shallow and deep corals In response to decreased light intensity, corals can either associated with different Symbiodinium genotypes, although deep present host (Brazeau et al., 2013) and/or symbiont genetic reefs host a huge biodiversity (Hovland, 2008). diversity (Lesser et al., 2010) as well as adaptations to grow under Here we compare the trophic and photosynthetic capacities low irradiance conditions. For example, in order to increase of two distinct holobionts of the scleractinian coral S. pistillata and optimize the light capture and rates of autotrophic carbon living in shallow and mesophotic reefs of Eilat (Red Sea). production at depth >30 m, corals undergo morphological S. pistillata, harbors clade A1 in shallow waters (<40 m) and changes (Mass et al., 2007; Einbinder et al., 2009; Nir et al., clade C in deeper environments (>40 m), forming two different 2011) as well as modifications in the photo-physiological traits of holobionts of the same (Byler et al., 2013). We measured, their symbionts (Wyman et al., 1987; Lesser et al., 2000, 2009). under the light conditions of surface and deep waters, the They also compensate their low photosynthate production by heterotrophic capacities of each holobiont, as well as their increasing the acquisition of heterotrophic nutrients (Muscatine rates of photosynthesis and acquisition of inorganic carbon 13 13 15 et al., 1989; Mass et al., 2007; Alamaru et al., 2009; Einbinder and nitrogen using stable isotopes [NaH CO3 ( C), NH4Cl 15 et al., 2009; Lesser et al., 2010; Crandall et al., 2016). Corals (NH4), and NaNO3 (NO3)]. Inorganic carbon and nitrogen can finally harbor different Symbiodinium clades depending on acquisition by the symbionts, together with host prey capture, the depth at which they thrive (Finney et al., 2010; Lesser provide direct measures to score nutritional plasticity of these et al., 2010). In the Caribbean, although some colonies can symbioses. Getting new insights into the trophic behavior and the present a mix of symbiont clades with a zonation pattern metabolic processes associated with mesophotic corals is essential (Kemp et al., 2015), Symbiodinium A and B are predominant to understand the ecology and biology of these unique habitats in tissue exposed to high irradiance (surface waters) while as well as the vertical connectivity existing between shallow and Symbiodinium C is dominant in shaded tissue of deep corals deep environments, which may be of great importance for the (Rowan and Knowlton, 1995; Rowan et al., 1997). In the northern future health and resilience of their associated ecosystems. Red Sea, Stylophora pistillata shifts from hosting clade A in shallow waters to clade C below 40 m depth (Mass et al., MATERIALS AND METHODS 2007; Winters et al., 2009). Although, numerous studies have now acknowledged the diversity and flexibility of Symbiodinium Biological Material and Experimental Setup under different light environments, the functional significance of Experiments were performed in November 2014 at the IUI, in these associations remains unexplored. It has been suggested that the Gulf of Eilat (Israel). While light conditions decreased from physiological diversification in Symbiodinium species can confer 200 to 400 µmol quanta m−2 s−1 at 5m depth to 20 to 40 µmol growth advantages to their associated coral host, by presenting quanta m−2 s−1 at 50m depth, the seawater temperature (24 ± different thermal and light tolerances as well as capacities in 0.5◦C) and nutrient concentrations remained stable throughout nutrient acquisition (Little et al., 2004; Abrego et al., 2008; Stat the investigated depth range (Bednarz et al., 2017). The above et al., 2008; Venn et al., 2008; Jones and Berkelmans, 2010; Lesser environmental data were provided by the Israel National et al., 2013). Among studies on the functional significance of the Monitoring Program at the Gulf of Eilat (http://www.iui-eilat. clades, most have focused on their capacity to tolerate thermal ac.il/Research/NMPMeteoData.aspx) and represented average stress (Berkelmans and van Oppen, 2006; Howells et al., 2012; values collected close to our study site. Ten mother colonies of Silverstein et al., 2015; Hume et al., 2016) while their nutritional the Red Sea coral S. pistillata were thus sampled directly on the capacities are still poorly known, although it has important reef, five colonies from 5 m depth and five others from 50 m physiological consequences for the host (Loram et al., 2007; Baker depth. Field collection of complied with a permit issued et al., 2013; Starzak et al., 2014; Leal et al., 2015). Baker et al. by the Israel Nature and Parks Authority (2015/40780). Colonies (2013) and Pernice et al. (2015) showed that the acquisition of were enclosed in different bags underwater, to keep track of nitrogen and carbon by Acroporidae species (Acropora tenuis each genotype, and brought back to the laboratory, located few and Isopora palifera, respectively) was clade—(Baker et al., 2013; meters from the reef, were they were properly identified. The Pernice et al., 2015) and temperature dependent (Baker et al., main Symbiodinium genotype hosted by each colony was checked 2013). Other studies on sea anemones also demonstrated that according to the protocol of Santos et al. (2002). As repeatedly Symbiodinium genotypic diversity affects the quantity and quality observed (Mass et al., 2007; Lampert-Karako et al., 2008; Winters of photosynthates translocated to the host (Loram et al., et al., 2009; Nir et al., 2014), the main clade associated to shallow 2007; Starzak et al., 2014; Leal et al., 2015), as well as the colonies was A1, while C1 and C3 (simplified to clades A and C

Frontiers in Marine Science | www.frontiersin.org 2 April 2017 | Volume 4 | Article 102 Ezzat et al. Nutrient Acquisition in Mesophotic Corals throughout the manuscript) populated the deep colonies (Table Fabricius (2000) and Gattuso and Jaubert (1990). P:R ratio, S5). Fifteen nubbins of about 3 cm long were then cut from which shows the autotrophic capability of an organism to self- each mother colony, tagged, and distributed in 4 open tanks maintenance was obtained with the following equation: P:R = for clade A and four other tanks for clade C. Aquarium system (PC × 12)/(RC × 24), where PC = Pg × 12/PQ and RC = R was set up in outdoor water tables, continuously supplied with × (12 × RQ). PC represents the amount of carbon acquired unfiltered oligotrophic seawater from the reef at a temperature of during photosynthesis; RC is the amount of carbon consumed 24◦C ± 0.5. Coral colonies were exposed to photon flux densities by respiration; 12 is the C molecular mass; PQ and RQ are (PFD) similar to those found at 50 and 5 m at the month of the photosynthetic and respiratory quotient respectively and collection (mean daily PFDs were equivalent to 13.8 mol photons vary according to the in situ light intensity experienced by m−2 d−1 for surface and 0.8 mol photons m−2 d−1 for deep the holobionts (Gattuso and Jaubert, 1990; Lesser, 2013). Net holobionts). Proper light intensity was calibrated by the useofa photosynthesis was considered to be efficient for 11 h light light meter (Li-Cor, LI-250A, USA) and UV-filters were disposed (length of the day in November in Eilat, http://www.iui-eilat. above aquaria to protect deep corals against UVR. Coral nubbins ac.il/NMP/Default.aspx) and respiration for 24 h. P:R ratio was were maintained for 2 days prior to the measurements described calculated for a period time of 24 h. below, which have been performed (i) at the “natural” light Additional nubbins (n = 5 per condition from 5 mother level of each population/holobiont (subsequently called “high- colonies) were used to assess the respiration rates of freshly light” for the shallow population and “low light” for the deep isolated symbionts. For this, tissues were extracted from live coral population) but also (ii) at the other corresponding light (low samples in 10 mL of FSW and centrifuged at the corresponding light for the shallow population and high light for the deep temperature. After centrifugation, pellets were re-suspended in one). This last measurement was performed to assess the ability 20 mL FSW and disposed for 20 min in aquaria before processing of each holobiont to quickly adjust its response to changes in to the respiration rate measurements as previously exposed. Data irradiance. were normalized to the surface area of each nubbin.

13 15 15 NaH CO3 and NH4Cl, NaNO3 Labeling Rates of Net Photosynthesis, Respiration, The following incubations were performed according to and Chlorophyll Content Measurements Tremblay et al. (2012) and Grover et al. (2002) (Figure 1). For Changes in oxygen production were monitored for 60 min on each holobiont (A and C), 40 coral nubbins (8 per colony) were 5 nubbins per holobiont (A and C; from 5 mother colonies) placed in individual beakers filled with 200 mL FSW. Beakers at the two mean irradiances corresponding to the surface and and corals were transferred back to the outdoor water tables; deep PFD levels: 350 µmol photons m−2 s−1 (shallow, High 20 beakers were incubated under ≪ low light ≫ while the light, HL) and 20 µmol photons m−2 s−1 (deep, Low light, LL), other 20 beakers were maintained under ≪ high light ≫ for respectively. Respiration rates (R) were assessed in the dark after 5h (T0), between 11 a.m. and 4 p.m., to cover the maximal the illumination period. Nubbins were individually placed in irradiances received at each depth. In addition, for each light 13 glass chambers, filled with a known volume of 0.45 µM filtered level, 10 beakers were enriched with 0.6 mM NaH CO3 (98 atom seawater (FSW) which were stirred using a magnetic stirrer. Glass %13C, #372382, Sigma-Aldrich, St-Louis, MO, USA) and 3 µM ◦ 15 15 chambers were maintained at 24 C by the use of a water bath NH4Cl (98 atom % N, #24858029, Sigma-Aldrich, St-Louis, 13 equipped with a PreSense optode (oxygen-sensitive minisensor) MO, USA) while 10 beakers received 0.6 mM NaH CO3 and 15 15 connected to the Oxy-4 (4 Channel oxygen meter, PreSense, 3 µM NaNO3 (98 atom % N, #24862444, Sigma-Aldrich, St- Regensburg, Germany). Optodes were calibrated against air- Louis, MO, USA). The concentrations were chosen to provide saturated and dinitrogen-saturated water for the 100 and 0% sufficient enrichment for further analysis. Additional nubbins oxygen, respectively. Estimations of net photosynthesis (Pn) at were incubated in non-enriched water as control samples LL and HL and respiration (R) were measured by regressing (natural isotopic abundance). At the end of the 5 h incubation, oxygen production against time. Gross photosynthesis (Pg) was half nubbins were sampled for T0 while the other half were assessed by adding R to Pn. At the end of each incubation, transferred into non-enriched seawater for one chase period ◦ ◦ coral nubbins were frozen at −20 C prior to the subsequent of 24h (T24). All nubbins were frozen at −20 C at the end determination of total chlorophyll content. Briefly, coral tissue of each time point (T0 and T24) before tissue extraction (air was removed from its skeleton in 10mL filtered seawater (FSW) pick), separation of the symbionts from the animal tissue by with an air-brush, homogenized with a potter grinder and centrifugation and freeze-drying of the extracts for conservation. transferred into a 15 mL tube before being centrifuged at 8,000 g The subsequent determination of %13C enrichment, for 10min at 4◦C. The pellet was re-suspended into 5 mL 100% %15N enrichment, C and N measurements in the algal and acetone and stored at 4◦C overnight. The next day, samples animal compartments were performed with a Delta plus Mass were centrifuged at 11,000g for 15min at 4◦C before assessing spectrometer (Thermofisher Scientific, Bremen, Germany) the chlorophyll content according to the protocol of Jeffrey and coupled to a C/N analyser (Flash EA, Thermofisher Scientific). Humphrey (1975). Data were normalized to the surface area The 15N or 13C enrichments were calculated from 15N-13C (cm2) of each nubbin using the wax-dipping technique (Davies, values obtained from samples exposed to 15N or 13C enriched 1989). Conversions of oxygen fluxes to carbon equivalents, based seawater and from control samples incubated without isotope on molar weights, were calculated according to Anthony and addition. From the difference between these two measurements,

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FIGURE 1 | Detailed table summarizing the experimental set up and the different parameters studied according to the number of nubbins used per timesteps [T0 (5 h incubation) and T24 (chase)]. Modified according to: http://ian.umces.edu/imagelibrary/displayimage-77-6640.html. the enrichment was calculated and considered significant when Furthermore, the amount of photosynthesized carbon it was at least three times higher than the standard deviation translocated from the symbionts to the host can be calculated obtained on control samples. From there, the nitrogen and using the following equation: carbon assimilation rates were calculated according to Montoya et al. (1996) based on the particulate nitrogen or carbon content Ts = Pc − ρs − Rs of the sample, the incubation time, the theoretical 15N and 13C enrichment of the incubation water at the beginning and the Where Pc is the total amount of autotrophic carbon produced skeletal surface area of the corals. by the symbionts; ρs is the carbon assimilation rate and Rs the The carbon incorporation rates in the symbionts and animal respiration rate of the symbionts. tissue was thus calculated according to Tremblay et al. (2012): Assimilation rates of ammonium and nitrate ρ were calculated according to Grover et al. (2002, 2003): (C − C ) ∗ M ∗ M ρ = meas nat s c N − N (Cinc − Cmeas) ∗ t + t ∗S mes nat 6 pulse chase ρ = ∗ Ms ∗ MN ∗ 10 (Nenr − Nmes) ∗ Tinc ∗ S Where ρ is the carbon incorporation rate in the algal or animal 13 Where N is the %15N measured in the sample; N is fraction, Cmeas and Cnat are the percentages of C measured in mes nat 13 the natural 15N abundance in control nubbin; N is the 15N the samples, Cinc is the percent C enrichment in the incubation enr medium, M is the mass of the freeze-dried sample (mg) and M enrichment of the incubation medium; Tinc is the incubation time s c 2 the mass of carbon per milligram of symbiont or animal tissues (h) and S is the surface area expressed in cm ; Ms is the total mass −1 2 of the sample (mg); M is the particulate nitrogen mass (mg). (µg mg ); S is the surface area of the nubbin (cm ) and Tpulse N For more detailed information regarding the calculations, and Tchase are the incubation time periods (h) in the light in please refer to Tremblay et al. (2012) and Grover et al. (2002). the enriched and non-enriched medium. Cinc varies during the pulse-chase period and can be calculated as following: Data were normalized to the coral surface area. Heterotrophic Feeding Cpulse ∗ tpulse + (Cchase ∗ tchase) Cinc = Feeding rates were determined at each light level (LL, HL) (tpusle + tchase) on 10 nubbins per holobiont (from 5 colonies) following Houlbrèque et al. (2004), by incubating individual fragments 13 Where Cpulse and Cchase are the percent C enrichment of in small 400 mL plexiglas chambers equipped with a motor- the enriched and non-enriched incubation media, respectively driven propeller to insure a proper flow speed of 4 cm s−1 as (Cchase = 1.1%). described in Ferrier-Pagès et al. (2010). Chambers were kept in

Frontiers in Marine Science | www.frontiersin.org 4 April 2017 | Volume 4 | Article 102 Ezzat et al. Nutrient Acquisition in Mesophotic Corals a water bath to maintain the temperature constant. After the full expansion of the polyps, the experiment started by delivering 1,000 Artemia salina nauplii L−1 (previously quantified using a Bogorov counting chamber). Artemia concentration was then determined in triplicate after 0, 15, 30, 45, and 60min and nauplii were directly replaced in the chamber after counting. Three additional chambers without coral were used to account for a possible decrease in nauplii concentration due to natural death. Feeding rates were calculated as the decrease in the number of preys during the incubation, and were normalized to the nubbin surface area. The concentration of A. salina nauplii used in this study (2,000 cells/L) is high compared to the zooplankton concentration usually measured in the reefs (7 cells/L). However, it is low compared to the concentration of nanoplankton prey (106–107 cells/L). It has only been used to assess the heterotrophic capacities of each coral population for a same amount of food.

Statistical Analysis Statistical analyses were performed using Statistica 10 (Statsoft, Chicago, IL). All data were expressed as mean ± standard error. Normality and homoscedasticity of the data residuals were tested using Kolmogorov–Smirnov (using Lilliefors corrections) and Levene tests. A one-way ANOVA was used to test the difference in chlorophyll content and respiration rates of the clade A and C-symbionts. Two-way ANOVAs were performed to test (i) the effects of the holobiont (A/C) and the light level (LL/HL) on the net and gross photosynthesis rates, the grazing rates and the P:R and (ii) to test the effect of holobiont (A/C) and fraction (host/symbiont) on the structural C:N. A factorial analysis of variance (ANOVA) was performed to test the effects of time (T0 and T24), light level, holobiont and fraction on the rates of carbon and nitrogen (NH4 and NO3) assimilation, the rates and percentage of photosynthate translocation, and the structural C and N contents (µmol C or N mg−1). Moreover, there was no significant difference between 13C measurements 15 15 obtained in presence of NH4 or NO3 either for A and C holobionts (ANOVA, p > 0.05), therefore data were pooled for analysis. Percentages of photosynthate translocation were log-transformed prior analyses. When there were significant differences, the analyses were followed by a posteriori testing (Tukey’s test). Finally, Pearson correlations were used to attest the relationships between (i) carbon fixation and translocation and (ii) feeding rates and NH4 or NO3 assimilation rates in the algal fraction after 5 h incubation. P-values were considered for p < α, α = 0.05.

RESULTS Physiological Measurements Chlorophyll concentration, which was only dependent on the in situ growth conditions of the holobionts (i.e., mainly light) was significantly lower in clade A than clade C-holobionts (Figure 2A; Table S1, ANOVA, p < 0.05). Net rates of −2 photosynthesis (Pn), significantly increased with light for both FIGURE 2 | (A) Total chlorophyll concentration [µg Chl (a+c2) cm ], (B) −2 −1 holobionts (Figure S1A; Tukey HSD, p < 0.001). In addition, Grazing rates (Number of prey cm mn ). Statistically significances are indicated in bold letters, p-values are considered for p < 0.05. while Pn was not different between holobionts under high light,

Frontiers in Marine Science | www.frontiersin.org 5 April 2017 | Volume 4 | Article 102 Ezzat et al. Nutrient Acquisition in Mesophotic Corals clade A-holobionts presented a lower Pn under low light (Figure S1A; Tukey HSD, p < 0.001). Respiration rates were significantly lower for clade C-holobionts (Figure S1B; Tukey HSD, p < 0.01). Therefore, clade A-holobionts reduced their P:R values with decreasing light intensities from 1.00 ± 0.08 to 0.11 ± 0.1 (Tukey HSD, p = 0.003) while the P:R ratio of clade C-holobionts remained unchanged (6.85 ± 1.5–4.3 ± 1.7 at high and low light respectively, Tukey HSD, p > 0.05), and was higher than the P:R of clade A-holobionts (Tukey HSD, p < 0.02). Feeding rates were significantly lower in clade A than clade C-holobionts, under their natural light level (Figure 2B; Table S1, ANOVA, p < 0.05 and p = 0.02). 13 NaH CO3 Labeling—Carbon Assimilation within the Symbiosis Carbon Acquisition and Allocation within the Symbiosis The carbon budget of each holobiont was similar in presence + − of NH4 and NO3 for each light level (ANOVA, p > 0.05). Therefore, data represent the mean and standard error of the two measurements. The amounts of carbon fixed by the symbionts and translocated to the host were significantly affected by both the light level and the clade hosted in each holobiont (Table S2; ANOVA, p < 0.002). Higher rates were indeed observed under high light for both holobionts (Figures 3A,B; Tukey HSD; p < 0.01). However, while clade A fixed and translocated 30% more carbon to the host than clade C under high light (Figures 3A,B; Table S2;Tukey HSD, p < 0.001), the reverse was observed under low light, especially for carbon translocation at T0 (Figure 3B; Tukey HSD, p < 0.05). The rates of carbon incorporation into the symbionts were generally significantly higher than those measured for the host (Figures 4A,B; Tukey HSD, p < 0.03). Higher rates were also generally observed under high light both in the animal and algal fractions of both holobionts (Figures 4A,B; Tukey HSD, p < 0.03). Ammonium Assimilation The assimilation of NH4 within the symbiosis was affected by both the clade present in each holobiont, the fraction (host/symbionts) considered and the length of the incubation (Table S3, ANOVA, p < 0.0002). Clade effect was very important as clade A-symbionts assimilated ca. 31 and 95% more NH4 than clade C-symbionts depending on the light level (Tukey HSD, p < 0.03). Clade A symbionts also retained 10–20 times more NH4 than their host (Figures 5A,B; Table S3; Tukey HSD, p < 0.0001), while clade C symbionts retained the same amount (Table S3; Tukey HSD, p > 0.05). The difference between T0 and T24 in the assimilation rates showed that clade A symbionts translocated to their host 6.2% of the NH4 assimilated while clade C symbionts translocated 47.5%. Nitrate Assimilation The assimilation of NO3 was affected by both the clade present in each holobiont, and the fraction (host/symbionts) considered FIGURE 3 | Rates of (A) Carbon fixation (µg C cm−2 h−1) and (B) Carbon (Table S3, ANOVA, p < 0.001). Higher assimilation rates were translocation (µg C cm−2 h−1) for clade A and C-holobionts according to the observed in the symbiont fraction compared to the host tissue light intensity. Statistically significances are indicated in bold letters, p-values are considered for p < 0.05. for both holobionts at any timesteps and light conditions

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FIGURE 4 | (A) Carbon incorporation rates (µg C cm−2 h−1) in symbionts 15 15 and (B) in host for both clade A and C-holobionts according to the different FIGURE 5 | Rates of (A) NH4 assimilation in symbionts and (B) NO3 −1 −2 fractions (algal and animal tissue), light intensities (LL/HL), and timesteps host (µg N h cm ) according to the different fractions (algal and animal tissue), light intensities (LL/HL), and timesteps (T ,T ). Statistically (T0,T24). Statistically significances are indicated in bold letters, p-values are 0 24 considered for p < 0.05. significances are indicated in bold letters, p-values are considered for p < 0.05.

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(Figures 6A,B; Table S3; Tukey HSD, p < 0.0001). Clade effect was also very important as clade A-symbionts assimilated ca. 38 and 67% more NO3 than clade C-symbionts depending on the fraction and time of incubation (Table S3, ANOVA, p < 0.001). In addition, the difference between T0 and T24 in the assimilation rates showed that clade A symbionts translocated 14.3% of the NO3 assimilated while clade C symbionts translocated 19.4%. An increase in the rate of NO3 assimilation was also observed between T0 and T24 in both clade C and clade A symbionts at any timesteps and light intensities (Table S3; Tukey HSD, p < 0.05), except for clade A symbionts exposed to low light (Tukey HSD, p > 0.05).

C:N Structural A significant interaction was observed between the fractions (host/symbionts) and the symbiont clade for the structural C:N ratio (Figure S5, Table S4, ANOVA, p < 0.001). The observed difference in C:N between holobionts were due to (1) lower carbon content (µmol C mg−1) of clade C holobionts, mainly in the host (Tukey HSD, p < 0.05; Figure S2); and to (2) lower nitrogen content (µmol N mg−1) of clade C- holobionts in both the host and symbionts (Figures S3, S4;Tukey HSD, p < 0.05). In addition, clade C symbionts showed higher C:N ratio compared to clade A symbionts (Table S4; Tukey HSD, p < 0.05).

Correlation between Studied Parameters A significant positive correlation was observed between the carbon fixation and translocation rates (Figure 7A; r = 0.918; d.f. = 18; p < 0.05). Significant negative correlations were observed between (a) the NH4 assimilation and feeding rates (Figure 7B, r = −0.737; d.f = 18; p < 0.05), (b) the NO3 assimilation and feeding rates (Figure 7C; r = −0.853; d.f = 18; p < 0.05).

DISCUSSION

This study assessed several aspects of the trophic ecology of the Red Sea coral S. pistillata associated with Symbiodinium clade A in shallow waters and clade C in deep environments (Winters et al., 2003; Mass et al., 2007) forming two different holobionts (Rohwer et al., 2002). The main finding is that clade C-holobionts presented a competitive advantage for the acquisition of carbon at low irradiance compared to clade A- holobionts. This observation is critical to explain the capacity of S. pistillata to colonize deep environments in the Red Sea. In addition, light was the primary factor that determined a positive relationship between the amount of carbon fixed in photosynthesis by the symbionts and the amount of carbon translocated to the host. Symbiodinium genotype was the primary factor influencing nitrogen assimilation rates, although light still played a relevant role in this relationship. Lastly, we observed a negative relationship between feeding rates and inorganic 15 FIGURE 6 | Rates of (A) NO3 assimilation in symbionts and (B) host (µgN nitrogen assimilation rates. Overall, our results suggest that −1 −2 h cm ) according to the different fractions (algal and animal tissue), light hosting different symbiont types allows the scleractinian species intensities (LL/HL), and timesteps (T0,T24). Statistically significances are S. pistillata to colonize both shallow and deep environments in indicated in bold letters, p-values are considered for p < 0.05. the Red Sea.

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−2 −1 −2 −1 15 15 FIGURE 7 | Relationships between (A) rates of carbon fixation (µg C cm h ) and translocation (µg C cm h ), feeding and (B) NH4 or (C) NO3 assimilation rates for clade A and C-holobionts according to the light intensity after 5 h incubation.

Acquisition of Inorganic Nitrogen are indicative of a lower metabolic activity, which in turn can The Symbiodinium genotype associated with shallow and deep lead to lower nitrogen requirements. On the contrary, survival in S. pistillata was one of the primary factors influencing the rates high light environments for clade A-holobionts can increase both of nitrogen assimilation and translocation. Clade A-holobionts their metabolic activity and their nitrogen demand to repair light- indeed presented higher NH4 and NO3 assimilation rates than damaged photosystems (Dubinsky and Jokiel, 1994; Dubinsky clade C-holobionts, independently of the irradiance received and Falkowski, 2011). during the incubation, and for the same nutrient history In addition, the higher assimilation of inorganic nitrogen by (comparable inorganic nutrient levels at 5 and 50 m depth in situ, clade A-holobiont may compensate for the lack of zooplankton, during the sampling month). Consistent with this finding, two which is known to be much less abundant in shallow waters recent studies (Baker et al., 2013; Pernice et al., 2015) found that compared to deep environments (Schmidt, 1973; Lesser et al., rates of nitrogen acquisition by Acroporidae species (A. tenuis 2009). Conversely, nitrogen can be acquired by clade C- and I. palifera, respectively) were also clade dependent. holobionts in deep waters through heterotrophy (Crandall et al., Light intensity/history was the second important factor 2016) or through dinitrogen fixation (Lesser et al., 2007). This explaining differences in nitrogen assimilation rates between is confirmed by their higher grazing rates compared to shallow the two holobionts. The importance of light was evidenced holobionts and by the inverse relationship between heterotrophy by the fact that (i) clade A-holobionts, which came from and inorganic nitrogen acquisition. This negative correlation the highly-lit shallow reef, and which presented a high suggests that nitrogen assimilation by the holobiont needs to metabolic activity, showed higher rates of both NO3 and be supported by heterotrophic feeding when the activity of NH4 assimilation than clade C-holobionts, sampled in deep the symbionts is reduced, either due to a reduction in light environment; (ii) NH4 assimilation by clade C-holobionts was availability or when the dominant symbiont type presents a higher at 350 µmol photons m−2 s−1 than at 20 µmol photons reduced ability for nitrogen assimilation. m−2 s−1. According to previous observations made on other Additionally, clade A and C-holobionts of S. pistillata had photosynthetic organisms (Canvin and Atkins, 1974; Amory unequal translocation rates and allocation patterns of nitrogen et al., 1991) or on coral symbionts in culture (Rodríguez-Román within the symbiosis, likely suggesting different needs of the and Iglesias-Prieto, 2005) and in hospite (Grover et al., 2002), symbionts and animal host in nitrogen. Consistent with previous light can have a direct effect on nitrogen assimilation; this results on many cnidarian species, clade A-holobionts showed later process is indeed closely dependent on the amount of a higher uptake of NH4 compared to NO3 (Wilkerson and electrons that can be supplied by the ferredoxin, through the Trench, 1986; Wilkerson and Kremer, 1993; Grover et al., 2002, linear electron flow between photosystems I and II. Light can 2003; Pernice et al., 2012), and very low translocation rates to also have an indirect effect on nitrogen assimilation, by changing the host. Clade A symbionts thus acted as a sink for nitrogen the metabolic activity of the holobionts. The low C and N within the symbiosis (Wilkerson and Kremer, 1993; Grover contents of clade C-holobionts compared to clade A-holobionts et al., 2002; Pernice et al., 2012; Kopp et al., 2013). In contrast,

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clade C-symbionts exhibited similar rates of NH4 and NO3 different light levels and clade genotypes, to better understand assimilation, and much higher translocation rates to the host. As this relationship. a consequence, nitrogen was evenly shared between the host and its symbionts. CONCLUSION

Carbon Budget Collectively, the results presented in this study indicate that The depths at which S. pistillata colonies were sampled (5 and the vertical distribution pattern of S. pistillata in the Red Sea, 50 m) account for extreme living conditions because holobionts and its ability to form mesophotic reefs, can be explained were exposed either to high irradiance (>350 µmoles photons by the presence of specific symbionts adapted to different − − − − m 2 s 1) or to <30 µmol photons m 2 s 1 (Cohen and light regimes. The differential use of light by clade A and C Dubinsky, 2015). By establishing the carbon budget of shallow symbionts constitutes an important axis for niche diversification, and deep holobionts of S. pistillata, this study first shows controlling the abundance and distribution of S. pistillata along that clade C-holobionts have a competitive advantage for the the depth gradient. Our results also show that the amount of solar acquisition of autotrophic carbon at low irradiance compared energy that the holobionts absorb drives their metabolic activity, to clade A-holobionts, and that each holobiont has a different nitrogen assimilation rates and needs as well as their structural environmental optimum along the irradiance gradient. This C:N content. study thus confirms previous observations, which concluded that S. pistillata acclimates to depth through photo-adaptive AUTHOR CONTRIBUTIONS mechanisms linked to the Symbiodinium genotype hosted at each depth distribution (Cohen and Dubinsky, 2015). Overall, many LE, CF, MF, and RG conceived the experiment. LE, CF, and MF coral species associate with more than one algal taxon, whose performed the experiments. LE and CF made the extractions. JM relative abundance has been correlated with irradiance gradients performed the mass spectrometry analyses. LE, CF, RG, and JM (Rowan and Knowlton, 1995; Rowan et al., 1997; Iglesias-Prieto analyzed the data. LE, CF, RG, MF, and JM wrote the manuscript. et al., 2004). These changes allow the host to extend its depth range while remaining ecologically dominant. Consistent also with previous observations on deep corals (McCloskey and ACKNOWLEDGMENTS Muscatine, 1984; Levy et al., 2006; Mass et al., 2007; Cohen and Dubinsky, 2015), low respiration rate was another adaptation of We would like to sincerely thank all the staff at the clade C-holobionts to their deep environment. This reduction IUI in Eilat (IL) as well as Dr. JO Irisson from the in respiratory needs maintained the P:R of clade C-holobionts, Observatoire Océanologique de Villefranche-sur-mer (LOV) measured at low light (4.3 ± 1.7), similar to the P:R of clade A- for his constructive comments and help with the statistical holobionts, measured under high light (1.00 ± 0.08), suggesting analyses, Vanessa Bednarz and Jérôme Durivault from the CSM. that the amount of photosynthates was sufficient to cover the Many thanks to Prof. Denis Allemand, Director of the Centre respiratory needs of the deep water colonies. All together, these Scientifique de Monaco for scientific support. Lastly, we would observations indicate a niche specialization among holobionts like to thank three reviewers for their constructive comments of S. pistillata, allowing a maximal acquisition and utilization of and time spent on revising our manuscript. This project was carbon throughout the depth profile. supported by the IUI, the Israel Science Foundation for MF, the The carbon budget also showed that carbon translocation CSM and the Institut Européen de la mer. Financial support to was closely related to carbon fixation in photosynthesis by LE was provided by the Centre Scientifique de Monaco. the symbionts and was therefore more closely related to light availability than to clade identity. This observation is in SUPPLEMENTARY MATERIAL agreement with a previous study performed on two scleractinian corals (Tremblay et al., 2015), but challenges the hypothesis The Supplementary Material for this article can be found that carbon translocation is clade dependent (Leal et al., 2015). online at: http://journal.frontiersin.org/article/10.3389/fmars. Further studies should be carried out, by taking into account 2017.00102/full#supplementary-material

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Frontiers in Marine Science | www.frontiersin.org 12 April 2017 | Volume 4 | Article 102